The Structural Requirements of Sterols for Membrane Function in Saccharomyces cerevisiae

Nes, W. David; Janssen, Giselle G.; Crumley, Farrist G.; Kalinowska, Małgorzata; Akihisa, Toshihiro

doi:10.1006/abbi.1993.1100

Cited by 107 publications

(72 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Whatever the origin of the effect there appears to be a difference in sensitivity between the ketosteroid reductases of different fungal species. Previous studies have indicated that the 14c~-methyl sterols preceeding this step, including eburicol, will not support growth [20] and this was consistent with our observations that a block to sterol biosynthesis was not associated with azole tolerance.…”

Section: Discussionsupporting

confidence: 92%

Resistant P45051A1 activity in azole antifungal tolerant Cryptococcus neoformans from AIDS patients

et al. 1995

View full text Add to dashboard Cite

Section: Discussionsupporting

confidence: 92%

Resistant P45051A1 activity in azole antifungal tolerant Cryptococcus neoformans from AIDS patients

et al. 1995

View full text Add to dashboard Cite

“…SMY10 (an SMY8 derivative expressing A. thaliana cycloartenol synthase (19) from the integrated yeast expression vector pRS305GAL) converts biosynthetic oxidosqualene to cycloartenol. We found that SMY10 requires ergosterol, like other lanosterol synthase mutants producing endogenous cycloartenol (28) or grown on exogenous cycloartenol (29). This is consistent with previous studies that showed low (30) (50) is alkylation followed by demethylation to cycloeucalenol, which is isomerized to obtusifoliol.…”

Section: Resultssupporting

confidence: 92%

“…If cycloartenol were the only sterol accumulating in the SMY10 host, isomerase expression would not restore ergosterol prototrophy. Fortunately, yeast at least partially demethylates cycloartenol at the 4-position (29,33). The yeast demethylating enzymes Erg25, Erg26, and Erg27 (34 -36) that normally metabolize lanosterol apparently have sufficiently broad substrate specificity to accept its isomer cycloartenol.…”

Section: Resultsmentioning

confidence: 99%

Functional Cloning of an Arabidopsis thalianacDNA Encoding Cycloeucalenol Cycloisomerase

Lovato¹,

Segura²,

Giner³

et al. 2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

Plants and certain protists use cycloeucalenol cycloisomerase (EC 5.5.1.9) to convert pentacyclic cyclopropyl sterols to conventional tetracyclic sterols. We used a novel complementation strategy to clone a cycloeucalenol cycloisomerase cDNA. Expressing an Arabidopsis thaliana cycloartenol synthase cDNA in a yeast lanosterol synthase mutant provided a sterol auxotroph that could be genetically complemented with the isomerase. We transformed this yeast strain with an Arabidopsis yeast expression library and selected sterol prototrophs to obtain a strain that accumulated biosynthetic ergosterol. The novel phenotype was conferred by an Arabidopsis cDNA that potentially encodes a 36-kDa protein.We expressed this cDNA (CPI1) in Escherichia coli and showed by gas chromatography-mass spectrometry that extracts from this strain isomerized cycloeucalenol to obtusifoliol in vitro. The cDNA will be useful for obtaining heterologously expressed protein for catalytic studies and elucidating the in vivo roles of cyclopropyl sterols.Even distantly diverged organisms generally biosynthesize shared molecules by such similar routes that bacteria and yeast have been suitable systems for deducing much of human metabolism. Sterol biosynthesis provides an exception to this trend. Eukaryotes use two distinct pathways ( Fig. 1) to make structurally similar sterols (1). Animals and fungi cyclize oxidosqualene to the tetracyclic ⌬ 8 -triterpene lanosterol (2), which they metabolize further to tetracyclic membrane sterols (3)(4)(5). Plants cyclize instead to the pentacyclic cycloartenol (6, 7) and consequently must isomerize the cyclopropane ring to form ⌬ 8 tetracycles en route to tetracyclic sterols. Why plants use this two-enzyme route (8) rather than cyclizing directly to tetracyclic sterols using lanosterol synthase remains unknown.Although the enzyme has not been purified to homogeneity, cycloeucalenol cycloisomerase (also known as cycloeucalenolobtusifoliol isomerase) activity has been observed directly in both dicots (9) and monocots (10). In addition, the presence of an isomerase has been demonstrated indirectly in diverse plants (11)(12)(13)(14) and the protozoan Acanthamoeba polyphaga (15) by identifying cyclopropyl sterols after treatment with isomerase inhibitors. In D 2 O, the isomerase adds a deuterium to C-19 and abstracts hydrogen from C-8 (16, 17). Neither ATP nor NADPH enhances activity (8), and acidic ring opening seems the most likely mechanism. Because the nonenzymatic acid-catalyzed isomerization (18) requires severe conditions (10% H 2 SO 4 in refluxing isopropyl alcohol for 24 h), it is likely that the enzyme employs a metal or an unusually acidic amino acid residue to open the ring.As a prelude to investigating its catalytic mechanism and biological function, we have cloned and heterologously expressed the Arabidopsis thaliana cDNA that encodes cycloeucalenol cycloisomerase. EXPERIMENTAL PROCEDURESCloning the Cycloeucalenol Cycloisomerase cDNA-The A. thaliana CAS1 1 cDNA (19) cloned into the integrative yeast exp...

show abstract

“…Variation in the conformation properties of the nucleus can affect the tilt of C-3 hydroxyl group and tilt of the 17(20)-bond sterol side chain. The possibility of cycloartenol to be "bent" is no longer credible, rather the sterol is thought to assume a planar structure with the side chain to the right when the substrate is bound to SMT or functioning as a membrane insert [3,30,31]. The global substrate features recognized by the SMT and turnover numbers developed from the Michaelis complex and related properties, including subunit architecture (4 identical subunits with native molecular weights between 161 kDa to 172 kDa), pH dependence (centered at pH 7.5) and activation energies (ca.…”

Section: Pro-smentioning

confidence: 99%

“…The ∆ 5 -phytosterols often constitute < 80% of the total sterol fraction of cells and accumulate in the plasma membrane [2][3][4][5]. The addition of a 24-alkyl group to the C 8 side chain of cholesterol can allow the side chain to sweep out a larger cone in the bulk lipids of the membrane than the cone swept out by a C 8 isooctyl side chain thereby providing greater potential of phytosterols to disrupt lipid-lipid interactions which in turn can affect fluidity of the lipid leaflet.…”

Section: Introductionmentioning

confidence: 99%

Mechanism-based Enzyme Inactivators of Phytosterol Biosynthesis

et al. 2004

View full text Add to dashboard Cite

Current progress on the mechanism and substrate recognition by sterol methyl transferase (SMT), the role of mechanism-based inactivators, other inhibitors of SMT action to probe catalysis and phytosterol synthesis is reported. SMT is a membrane-bound enzyme which catalyzes the coupled C-methylation-deprotonation reaction of sterol acceptor molecules generating the 24-alkyl sterol side chains of fungal ergosterol and plant sitosterol. This C-methylation step can be rate-limiting in the post-lanosterol (fungal) or post-cycloartenol (plant) pathways. A series of sterol analogs designed to impair SMT activity irreversibly have provided deep insight into the C-methylation reaction and topography of the SMT active site and as reviewed provide leads for the development of antifungal agents.

show abstract

The Structural Requirements of Sterols for Membrane Function in Saccharomyces cerevisiae

Cited by 107 publications

References 0 publications

Resistant P45051A1 activity in azole antifungal tolerant Cryptococcus neoformans from AIDS patients

Resistant P45051A1 activity in azole antifungal tolerant Cryptococcus neoformans from AIDS patients

Functional Cloning of an Arabidopsis thalianacDNA Encoding Cycloeucalenol Cycloisomerase

Mechanism-based Enzyme Inactivators of Phytosterol Biosynthesis

Contact Info

Product

Resources

About